Pinot blanc and Pinot gris arose as independent somatic mutations of Pinot noir

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RESEARCH PAPER

In Posidonia oceanica cadmium induces changes in DNA

methylation and chromatin patterning

Maria Greco, Adriana Chiappetta, Leonardo Bruno and Maria Beatrice Bitonti*

Department of Ecology, University of Calabria, Laboratory of Plant Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende, Cosenza, Italy

* To whom correspondence should be addressed. E-mail: b.bitonti@unical.it Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011

Abstract

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE, DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.

Introduction

In the Mediterranean coastal ecosystem, the endemic seagrass Posidonia oceanica (L.) Delile plays a relevant role by ensuring primary production, water oxygenation and provides niches for some animals, besides counteracting coastal erosion through its widespread meadows (Ott, 1980; Piazzi et al., 1999; Alcoverro et al., 2001). There is also considerable evidence that P. oceanica plants are able to absorb and accumulate metals from sediments (Sanchiz et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus influencing metal bioavailability in the marine ecosystem. For this reason, this seagrass is widely considered to be a metal bioindicator species (Maserti et al., 1988; Pergent et al., 1995; Lafabrie et al., 2007). Cd is one of most widespread heavy metals in both terrestrial and marine environments.

Although not essential for plant growth, in terrestrial plants, Cd is readily absorbed by roots and translocated into aerial organs while, in acquatic plants, it is directly taken up by leaves. In plants, Cd absorption induces complex changes at the genetic, biochemical and physiological levels which ultimately account for its toxicity (Valle and Ulmer, 1972; Sanit_{z di Toppi and Gabrielli, 1999; Benavides et al., 2005;} Weber et al., 2006; Liu et al., 2008). The most obvious symptom of Cd toxicity is a reduction in plant growth due to an inhibition of photosynthesis, respiration, and nitrogen metabolism, as well as a reduction in water and mineral uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000; Shukla et al., 2003; Sobkowiak and Deckert, 2003).

At the genetic level, in both animals and plants, Cd can induce chromosomal aberrations, abnormalities in

ª 2011 The Author(s).

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ReseaRch PaPeR

Pinot blanc and Pinot gris arose as independent somatic

mutations of Pinot noir

Silvia Vezzulli1,_{*, Lorena Leonardelli}1_{, Umberto Malossini}2_{, Marco Stefanini}1_{, Riccardo Velasco}1_and Claudio Moser1

1 _{Research and Innovation Centre, Fondazione Edmund Mach, via E. Mach 1, 38010 San Michele a/Adige (TN), Italy} 2 _{Consulting and Services Centre, Fondazione Edmund Mach, via E. Mach 1, 38010 San Michele a/Adige (TN), Italy} *To whom correspondence should be addressed. E-mail: silvia.vezzulli@fmach.it

Received 3 July 2012; Revised 21 September 2012; Accepted 24 September 2012

Abstract

Somatic mutation is a natural mechanism which allows plant growers to develop new cultivars. As a source of varia-tion within a uniform genetic background, it also represents an ideal tool for studying the genetic make-up of impor-tant traits and for establishing gene functions. Layer-specific molecular characterization of the Pinot family of grape cultivars was conducted to provide an evolutionary explanation for the somatic mutations that have affected the locus of berry colour. Through the study of the structural dynamics along chromosome 2, a very large deletion present in a single Pinot gris cell layer was identified and characterized. This mutation reveals that Pinot gris and Pinot blanc arose independently from the ancestral Pinot noir, suggesting a novel parallel evolutionary model. This proposed ‘Pinot-model’ represents a breakthrough towards the full understanding of the mechanisms behind the formation of white, grey, red, and pink grape cultivars, and eventually of their specific enological aptitude.

Key words: Berry colour, grapevine, layer, molecular characterization, SSRs and SNPs, Vitis vinifera.

Introduction

As a source of variation within a uniform genetic background, somatic mutation in plants is of theoretical and practical interest. A great deal of the genetic variation exploited in plant breed-ing comes from somatic variations that naturally arise in many plant groups and are used by growers in developing new cul-tivars that are agronomically and/or commercially superior to the parent stock. Indeed, many of these mutants have become a major source of horticultural staple crops (e.g. potato), fruit trees (Citrus spp., peach, banana, etc.), and ornamental plants

(rose, dahlia, chrisanthemum, etc.) (Shamel and Pomeroy, 1936).

It should be emphasized that the great majority of somatic muta-tions probably go unnoticed because they do not visibly affect easily detectable characteristics such as colour and growth habit

of the plant (Hartmann and Kester, 1975).

Unlike higher animals, in higher plants somatic mutations can enter the germline and be transmitted to the progeny. Whether or

not a somatic cell enters the germline depends on its localization

in a defined cell layer (L) in the shoot apex (D’Amato, 1997).

In dicots, gametes arise from the L2 lineage (Marcotigiano and Bernatzky, 1995). Indeed, most dicots have stratified api-cal meristems containing up to three layers of dividing cells, the combination of which gives rise to different plant tissues (Neilson-Jones, 1969). Mutations could be present in the entire meristem (bud sports) or only a portion (chimeras). Chimeras are composed of two or more genetically distinct tissue layers that

grow adjacent to one another (Dermen, 1960). Even if a somatic

mutation is not incorporated into a cell line that differentiates into gametes, it can still be perpetuated by asexual reproduc-tion. This makes somatic mutation a valuable source of heritable variation especially in plants which are multiplied via vegetative propagation, including grapevine (Vitis vinifera L.). The grow-ing scientific interest in the phenomenon of somatic mutation

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

at Istituto Agrario di S.Michele all'Adige on May 13, 2013

http://jxb.oxfordjournals.org/

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in grapevine is demonstrated by a list of recent publications. To

date, in addition to anthocyanin production (Kobayashi et al.,

2004; Walker et al., 2006), somatic genetic variation has been successfully used to identify genes involved in gibberellic acid

signalling (Pinot meunier; Boss and Thomas, 2002), early berry

morphogenesis (Fernandez et al., 2006), and flower development

(Chatelet et al., 2007; Fernandez et al., 2010).

Among grapevine somatic variants, those affecting fruit col-our are probably the most studied since they occur in several

cul-tivars (Galet, 2000). Grape berry colour is due to the presence of

a single pigment family, the anthocyanins, which vary greatly in concentration and composition depending on the grape cultivar (Mattivi et al., 2006). In many plants, anthocyanin biosynthesis is controlled by regulatory genes belonging to the Myb family of

transcription factors (Koes et al., 2005). Two Myb-related

tran-scription factor genes, VvMybA1 and VvMybA2, regulate antho-cyanin biosynthesis in V. vinifera grapes. Inactivation of these two functional genes, through the insertion of the Gret1 retro-transposon in the VvMybA1 promoter and through a non-syn-onymous single nucleotide polymorphism (SNP) present in the

VvMybA2 coding region, gives rise to a white berry phenotype

(Kobayashi et al., 2004, 2005; Walker et al., 2007). Recently, several genetic and genomic studies revealed that the colour locus is a cluster of four Myb-like genes located on chromosome 2 (Walker et al., 2006; Matus et al., 2008; Azuma et al., 2009;

Fournier-Level et al., 2009).

Since Pinot is a founder variety, it had several chances to

undergo somatic mutations (Hocquigny et al., 2004; Lacombe

et al., 2011). Most of these affected the ancestral black berry

col-our, and gave rise to cultivars such as in Pinot blanc and Pinot gris. The most established evolutionary model is that Pinot blanc arose from Pinot gris which arose from Pinot noir, even if the relationship between Pinot blanc and Pinot gris has not yet been

fully explored (Viala and Vermorel, 1909; Pelsy, 2010). Pinot

gris is reported to be a periclinal chimera of Pinot noir, but also in this case the exact nature of the genetic modification remains

to be determined (Franks et al., 2002; Hocquigny et al., 2004).

Chimerism in grapevine was first observed in the cell layers of shoot apical meristems (SAMs) as variability in the level of ploidy (cytochimeras). These studies demonstrated that the grapevine apical meristem is composed of only two distinct tissue layers,

L1 and L2 (Einset and Pratt, 1954; Thompson and Olmo, 1963).

Investigation of somatic mutations affecting the fruit col-our locus will improve knowledge of the genetics of grey- and white-skinned cultivars as well as promote understanding of the evolutionary events behind their origin. Here, a layer-specific structural analysis at both the genome-wide and berry colour locus level in several Pinot noir, Pinot gris, and Pinot blanc clones along with their naturally derived chimeras or sports is reported. The findings provide experimental evidence of a novel evolutionary model for the Pinot family of grape cultivars.

Materials and methods

Plant material and genomic DNA extraction

A set of 29 clones belonging to four V. vinifera cultivars were studied: (i) four clones of Pinot noir, including the sequenced Pinot noir clone ENTAV 115; (ii) 13 clones of Pinot gris, of which eight produced only

grey-skinned berries, two bore wholly white-skinned berries, one pro-duced berry sectorial chimeras and wholly white-skinned berry clusters, one produced bud sports (wholly mutated buds), and one displayed sec-torial chimeras, wholly white-skinned berries, and wholly mutated buds; (iii) 10 clones of Pinot blanc; and (iv) two clones of Pinot meunier, as a somatic variant for an additional trait. The studied clones were regis-tered by several European institutes and housed at Fondazione Edmund Mach (Italy), at the Laimburg Research Centre (Italy), and at the germ-plasm national repository of CRA-VIT (Italy) (Fig. 1; Supplementary Table S1 available at JXB online). For each clone, genomic DNA was isolated from 100–200 mg of young leaf and/or berry skin (L1+L2) and 300–500 mg of berry flesh and/or root (L2). To avoid contamination, berry flesh samples were isolated by dissecting the cells between the berry skin and the tissue surrounding seeds. After freeze-drying, leaf and root material was ground using an MM 300 Mixer Mill (Retsch Inc., Haan, Germany) and DNA extraction was performed using the DNeasy 96 Plant Mini Kit (Qiagen, Hilden, Germany) according to the manu-facturer’s protocol. After liquid nitrogen grinding, DNA isolation from berry skin and flesh powders was carried out according to Lodhi et al. (1994). A total of 117 tissue-specific DNA samples were obtained and divided into a core set of 40 samples, representing 10 wild-type clones (four tissues) of the considered Pinot cultivars, and an extended set of 77 samples, containing derived chimeras and sports, and additional wild-type Pinot gris and Pinot blanc (Supplementary Table S1). It is impor-tant to emphasize that in each analysis the genetic make-up of L1 was derived from the difference between the L1+L2 (leaf, berry skin) and L2 (berry flesh, root) profiles.

SSR analysis

For each simple sequence repeat (SSR) locus, primer sequence infor-mation, annealing temperature (Ta), and type of Taq polymerase are reported in Supplementary Table S2 at JXB online. Genomic DNA was amplified by PCR according to the following conditions: 20 ng of DNA template, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 mM fluorophore-labelled forward and reverse primer, 0.25 U of Taq DNA polymerase, and milliQ water to 12.5 ml PCR final volume. PCR ther-mocycling was performed with a 5/10 min initial denaturation/activation step, followed by 35 cycles at 94 °C for 60 s, Ta for 45 s, and 72 °C for 90 s, with a final extension step of 7 min at 72 °C. The presence of PCR products was assessed by electrophoresis with a 1.5% agarose gel and quantified by comparison with a Mass ruler DNA ladder mix (Thermo Fischer Scientific, Waltham, MA, USA). Capillary electrophoresis of PCR products was carried out on a 3730xl Genetic Analyzer (Life Tech, Foster City, CA, USA). Allele identification was performed by using GeneMapper v4.0 software (Life Tech).

SNPlex assay and data analysis

The SNPlex assay (Life Tech; Tobler et al., 2005) was carried out on ~40 ng of genomic DNA fragmented by heat shock. Ten SNP sets, for a total of 430 electronic SNPs (eSNPs), were chosen based on the number of validated SNPs which were first identified in Pinot noir clone ENTAV 115 (Pindo et al., 2008; Vezzulli et al., 2008). SNPlex assay, capillary electrophoresis, and genotype analysis were carried out according to

Pindo et al. (2008).

Sequencing analysis

The overall SNP regions, the VvMyb1, VvMyb2, and VvMyb3 promoter regions (encompassing the first exon), and the 3’ untranslated region (UTR), along with the VvMyb4 gene (exons and introns), were sequenced as described below. Flanking regions, between 300 bp and 400 bp long, were sequenced for each of the informative target SNPs in order to con-firm the SNPlex results and to discover all possible polymorphic sites. PCR primers of VvMyb genes were derived from Fournier-Level et al. (2009), while PCR primers for SNP (non-coding) and gene (coding) regions were designed based on the Pinot noir and the PN40024 genomic sequence, respectively (Jaillon et al., 2007; Velasco et al., 2007), using

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the Primer3 software (Rozen and Skaletski, 2000) (Supplementary Table S3 at JXB online). PCRs were assembled using the following con-ditions: 10–20 ng of genomic DNA, 1× PCR buffer (Life Tech), 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 µM of each primer, 1 U of Taq DNA polymerase (Life Tech), and water to a final volume of 12.5 µl. The DNA amplifications were performed using a 5 min initial denaturation step, followed by 30 cycles at 94 °C for 30 s, Ta for 30 s, and 72 °C for 60 s, with a final extension step of 10 min at 72 °C. The presence of PCR products was assessed by electrophoresis in a 1.5% agarose gel. In order to remove unincorporated dNTPs and primers, amplicons were enzymatically purified with exonuclease-phosphatase (ExoSAP-IT, GE Healthcare, Piscataway, NJ, USA). Sequencing reactions, post-sequencing purification, capillary electrophoresis, and polymorphism detection were carried out as reported by Vezzulli et al. (2008). The amplicon sequences were searched by BLAST-N (Altschul et al., 1990) against the genome assembly of both the highly heterozygous Pinot noir ENTAV 115 genotype (Velasco et al., 2007) and the near-homozygous PN40024 line (Jaillon et al., 2007).

Results

Layer-specific genome-wide analysis

Nine reference SSR markers (Supplementary Table S2 at JXB

online) were used to confirm that the Pinot core set used in this study was true to type. According to this analysis, Pinot noir, Pinot gris, and Pinot blanc share the same genotype (Supplementary Table S4a), as reported by Regner et al. (2000). In contrast, being a grape chimera, Pinot meunier showed a

tri-allelic profile at the reference locus VVS2 (Thomas and Scott,

1993) in agreement with Franks et al. (2002) (Supplementary

Table S4a). In order to test for any additional chimeric genetic presence in the core set, these samples were also analysed with

six SSR markers prone to triallelism (Supplementary Table S2).

The triallelic SSR analysis did not reveal any chimeric state and

confirmed that the Pinot family members share a uniform genetic

background (Supplementary Table S4b).

Of the 430 eSNPs analysed on the Pinot core set in the SNPlex experiment, 284 satisfied the quality value, with a mean call rate of 98% per SNPset, and therefore were considered successful, while the remaining 146 were not included in the genetic analy-sis. Further testing of the 284 eSNPs showed that 220 were true positive (heterozygous) Pinot noir SNPs, while 64 were false pos-itive. As anticipated by the SSR analysis, Pinot noir, Pinot blanc, and Pinot meunier share a highly uniform genetic background, since no differences were observed at 215 out of 220 SNP loci. Interestingly, unlike Pinot noir and Pinot blanc, Pinot gris clones had five SNPs (SNP4045, SNP7234, SNP4071, SNP7054, and SNP6166) that were not heterozygous in both L1+L2 and pure L2 tissues. Therefore, these SNPlex results did not allow dis-crimination of either clones or layers within each cultivar. Due to the already reported detection limits, such as preferential

anneal-ing, of this genotyping system (Pindo et al., 2008; Vezzulli et al.,

2008), the derived SNP information was validated by sequencing.

The sequencing analysis of the Pinot core set at the five informative SNP loci (SNP4045, SNP7234, SNP4071, SNP7054, and SNP6166) mostly confirmed the genotype identified by SNPlex analysis. In fact, the SNPlex results were validated in all Pinot clones except for those of Pinot gris. Although appear-ing homozygous in each layer of the Pinot gris clones follow-ing SNPlex analysis, the five SNP regions had a layer-specific genetic make-up according to the sequencing analysis. In par-ticular, while being heterozygous in the L1+L2-derived tissues, they were homozygous-like in the L2-derived tissues. However, in the heterozygous profiles, one allele was always under-repre-sented, compared with the reference heterozygous profile of Pinot

noir (Fig. 2). In addition to the five SNPs targeted by SNPlex

Fig. 1. Mosaic of berry colour variations in the studied Pinot somatic variants. (a) A berry cluster of Pinot noir; (b) berry clusters of Pinot blanc; (c) a berry cluster of wild-type Pinot gris and wholly skinned berry clusters from the same vine of Pinot gris; (d) wholly white-skinned berries of Pinot gris; and (e) a berry sectorial chimera of Pinot gris.

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assay, the resequencing analysis allowed for the detection of 15 point mutations along the five corresponding SNP regions in the

L1+L2-derived tissues of the Pinot cultivars (Supplementary

Table S5 at JXB online).

A BLAST-N search against the genome sequence of both the

highly heterozygous Pinot noir ENTAV 115 genotype (Velasco

et al., 2007) and the near-homozygous PN40024 line (Jaillon

et al., 2007) located these five informative SNP regions along

chromosome 2, near the Myb-related genes responsible for anthocyanin synthesis in grape.

Layer-specific structural analysis of the colour locus

To investigate further the genetic structure of this section of chromosome 2, the four Myb-related genes were amplified and

sequenced in the Pinot core set (Supplementary Table S3 at JXB

online). The main results (Fig. 3; Supplementary Table S5, S6)

can be summarized as: (i) the VvMybA4 gene and the 3’UTR of VvMybA2 did not contain any polymorphisms in any Pinot genotype or layer; (ii) the VvMybA2 promoter as well as the

VvMybA1 3’UTR and VvMybA1 first exon were heterozygous in

both Pinot noir layers and in the L1 of Pinot gris, while showing a homozygous-like genetic make-up in the L2 layer of Pinot gris and in both Pinot blanc cell layers; (iii) the VvMybA1 promoter contained the Gret (non-functional) and non-Gret (functional) alleles in both Pinot noir cell layers and in the L1 cell layer of Pinot gris, while only the Gret (non-functional) allele was pre-sent in the L2 layer of Pinot gris and in both Pinot blanc cell lay-ers; and (iv) primers designed within the VvMybA3 promoter and

3’UTR did not amplify a single target region, although different PCR conditions were applied on the Pinot core set.

In order to confirm the presence and to assess the extent of the homozygous-like region in the L2 layer of Pinot gris and in both cell layers of Pinot blanc, seven SSR markers and four SNP

regions (Supplementary Table S2, S3 at JXB online) inserted

among the Myb-related gene and the five informative SNP regions were genotyped in the Pinot core set. The VVNTM1 and VVNTM2 SSR markers were homozygous-like in the L2 layer of Pinot gris and in both Pinot blanc cell layers, but heterozy-gous in both Pinot noir cell layers and in the L1 layer of Pinot gris. Three SSR markers (VVNTM3, VVNTM5, and VVIU20) and four SNP regions (SNP7253, SNP0060, SNP8066, and SNP7002) were heterozygous in both layers of Pinot noir and Pinot blanc, and in the L1 layer of Pinot gris, but homozygous in the L2 cell layer of Pinot gris. VVNTM4 and VVNTM6 SSR markers did not display any polymorphisms in the Pinot

geno-types and cell layers (Fig. 3; Supplementary Table S5, S6).

To extend the genetic and structural characterization upstream and downstream of the colour locus region, eight additional loci were analysed in the Pinot core set. In particular, four SSR markers were

genotyped and four SNP regions were sequenced (Supplementary

Table S2, S3 at JXB online). Out of the seven loci upstream of the SNP4045 region, the SC08_0146_026 SSR was not polymorphic, whereas the SC08_0146_010 SSR was heterozygous in each Pinot genotype and cell layer, as were the more upstream loci, namely SNP6097, SNP6003, VMC6B11, SNP4165, and SNP6139. The VMC7G3 SSR locus downstream of the SNP6166 region showed a heterozygous profile in both layers of Pinot noir and Pinot blanc,

Fig. 2. Comparison of the electropherograms obtained by resequencing the SNP6166 region among the studied Pinot colour somatic variants. Differently coloured circles highlight the point mutations Y (C/T) and M (A/C) in leaf (L), berry skin (S), berry flesh (F), and root (R) of Pinot noir (PN), Pinot gris (PG), and Pinot blanc (PB). Allelotyping and peak height are depicted. In particular, (a) shows the heterozygous state with an under-represented allele in L1+L2-derived tissues of PG, while (b) reveals the homozygous-like state in pure L2-derived tissues of PG. (c) and (d) show the fully heterozygous state in both L1+L2-derived and pure L2-derived tissues of PN and PB.

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and in the L1 layer of Pinot gris, while this marker was

homozy-gous-like in the L2 layer of Pinot gris (Fig. 3; Supplementary Table

S5, S6). To map the 5’ border of the homozygous-like region in the L2 layer of Pinot gris more closely, six additional gene regions between the SC08_0146_010 and SNP4045 markers were

ana-lysed in the Pinot core set (Supplementary Table S3). Within this

gap, the SV02, SV06, and SV09 regions were heterozygous, while SV08 was not polymorphic in any Pinot genotype or cell layer. SV10 and SV12 showed a heterozygous profile in both layers of Pinot noir and Pinot blanc, and in the L1 layer of Pinot gris, while

both markers were homozygous-like in the L2 of Pinot gris (Fig. 3;

Supplementary Table S5, S6).

In conclusion, this work, aimed at the confirmation and delimi-tation of the homozygous-like region in the L2 cell layer of Pinot gris and in both layers of Pinot blanc, led to the identification of 14 informative loci, in addition to the five SNP regions

previ-ously targeted by SNPlex analysis (Supplementary Table S5 at

JXB online). In cultivars derived from asexual (vegetative)

repro-duction, such as the Pinot cultivars, this homozygous-like profile can only be ascribed to the presence of a deletion. Therefore, the ‘homozygous-like’ term corresponds to the genetic term ‘hemizygous’, which refers to the presence of a null allele. Thus, this study revealed that the Pinot gris deletion affects the L2 cell

layer only and extends for a minimum of 4202 kb and a maxi-mum of 4350 kb, while the Pinot blanc deletion occurs in both cell layers and ranges from 100 kb to 179 kb. These results are consistent with recent non-cell layer-specific findings, in which both Pinot blanc and Pinot gris were shown to be carrying a

par-tially deleted allele at the berry colour locus (Hocquigny et al.,

2004; Walker et al., 2006; Yakushiji et al., 2006).

In order to ascertain if this genetic structure exists in clones that originate from different viticultural areas, 13 of the 36

markers along chromosome 2 (Supplementary Table S6 at JXB

online)-chosen as informative, well scattered, and easy to score-were screened on an extended set of plants consisting of mutants and additional wild-type Pinot blanc and Pinot gris. Of these, the eight Pinot blanc clones showed the same L1 and L2 cell layer genetic profiles as the core set Pinot blanc samples, and the six grey-berried clones of Pinot gris also confirmed the L1 and L2 layer genetic profiles of the core set Pinot gris samples. Moreover, additional Pinot gris clones bearing from wholly white-skinned berries to wholly mutated buds were shown to share the genetic make-up of the L2 cell layer of the wild-type Pinot gris clones. However, the grey skin sectors of sectorial chimeras possessed the L1 and L2 cell layer genetic profiles of the wild-type Pinot

gris clones (Table 1).

Fig. 3. Chromosome 2 structure in the Pinot core set. A snapshot of the chromosome 2 region depicts different molecular markers showing either a heterozygous genetic state (he) or a heterozygous deletion (hd) in leaf (L), berry skin (S), berry flesh (F), and root (R) of Pinot noir (PN), Pinot gris (PG), and Pinot blanc (PB) registered clones.

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Table 1. Berry colour locus structure in the extended set of Pinot gris (PG) , along with its naturally derived mutants, and Pinot blanc (PB) clones, based on the analysis of 13 informative and well-scattered loci at the layer-specific level.

Cv Clone State Berry description Tissue

code SV 9 SV 10 SNP 4045 VVN TM1 VVN TM2 VvMybA1 promoter VVN TM3 SNP 7234 VVN TM5 SNP 7054 VVI U20 SNP 6166 VMC 7G3

PG SMA505 MUT L he hd hd hd hd Gret/– hd hd hd hd hd hd hd

MUT Wholly mutated buds S he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly mutated buds F he hd hd hd hd Gret/– hd hd hd hd hd hd hd PG SMA514 MUT Wholly white-skinned berries S he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly white-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

PG 49/207FR WT L he he he he he Gret/non-Gret he he he he he he he

WT Grey-skinned berries S he he he he he Gret/non-Gret he he he he he he he WT Grey-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

PG CL52 WT L he he he he he Gret/non-Gret he he he he he he he

PG CL53 WT L he he he he he Gret/non-Gret he he he he he he he

PG 457 WT L he he he he he Gret/non-Gret he he he he he he he

PG FEDIT13 WT L he he he he he Gret/non-Gret he he he he he he he

PG R6 WT L he he he he he Gret/non-Gret he he he he he he he

PG VCR5 WT L he he he he he Gret/non-Gret he he he he he he he

PG 10–05 WT L he he he he he Gret/non-Gret he he he he he he he

WT Grey-skinned berries S he he he he he Gret/non-Gret he he he he he he he WT Grey-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly white-skinned berries S he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly white-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

PG 10–10 WT L he he he he he Gret/non-Gret he he he he he he he

WT Grey-skinned berries S he he he he he Gret/non-Gret he he he he he he he WT Grey-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd PG 513 WT Grey-skinned berries S he he he he he Gret/non-Gret he he he he he he he WT Grey-skinned berries F he he he he he Gret/non-Gret he he he he he he he MUT_

wt

Grey sector of sectorial chimera

S he he he he he Gret/non-Gret he he he he he he he MUT_

mut

White sector of sectorial chimera

S he hd hd hd hd Gret/– hd hd hd hd hd hd hd

MUT Sectorial chimera F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

MUT L he hd hd hd hd Gret/– hd hd hd hd hd hd hd

MUT Wholly white-skinned berry clusters

F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

PG 516 WT L he he he he he Gret/non-Gret he he he he he he he

WT Grey-skinned berries S he he he he he Gret/non-Gret he he he he he he he WT Grey-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT_

wt

Grey sector of sectorial chimera

S he he he he he Gret/non-Gret he he he he he he he MUT_

mut

White sector of sectorial chimera

MUT Sectorial chimera F he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly white-skinned berries S he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly white-skinned berries F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

(Continued)

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Discussion

The layer-specific analysis of somatic variation

Somatic variation is important for genetic improvement in fruit trees, such as grape, where vegetative reproduction is used to propagate interesting new phenotypes, appearing as spontaneous sports. In spite of its relevance, little is known about the genetic and epigenetic mechanisms causing this variation, which might be in a chimeric state. For this reason, to study the Pinot somatic mutations, a layer-specific approach at a genomic scale was cho-sen, which turned out to be crucial.

In a first attempt to assess the impact of the chimerism on the intravarietal genetic diversity in the Pinot family of grape cul-tivars, a collection of clones belonging to the Pinot noir, Pinot gris, Pinot blanc, and Pinot meunier cultivars was analysed at a cell layer level with SSRs prone to triallelism. Given the intrin-sic feature of SSRs in producing stutter bands, the layer-specific analysis prevented the identification of false-positive results. This study confirmed that the genetic background of the Pinot family members is highly uniform and showed the presence of the VVS2 microsatellite mutation in all tested Pinot

meu-nier clones, in agreement with previous reports (Franks et al.,

2002; Hocquigny et al., 2004). Unlike the present analysis, this mutation was also found in Pinot gris clones of different origin

(Hocquigny et al., 2004). This suggests that the VVS2 locus is highly plastic and that this mutation most probably occurred independently after the Pinot gris and Pinot meunier divergence, namely after the respective mutations causing the partial loss of

berry colour (Walker et al., 2007) and the hairy leaf phenotype

(Boss and Thomas, 2002). This latter hypothesis is in contrast

to the conclusion by Hocquigny et al. (2004) that Pinot

meu-nier derives from Pinot gris, based on their non-layer-specific and non-genome-extensive approach. This discrepancy leads to a further hypothesis based on which these multiple mutations are recurrent. Therefore, it is not possible to speculate on which mutation occurred earlier than the other one.

Pinot cultivars show primitive morphological features analo-gous to those of the wild-type V. vinifera sbs. sylvestris, and are

thus considered archaic cultivars (Levadoux, 1956). As one of

the founder varieties and cultivated worldwide, Pinot has accu-mulated mutations leading to peculiar agronomical and enologi-cal aptitudes. The most relevant is the berry skin colour mutation leading to Pinot cultivars with different pigmentation. Berry skin colour mutants exist for a range of grape varieties (e.g. Grenache, Traminer, etc.) as well as for other fruit species (e.g. apple, pear, etc.) (Germplasm Resources Information Network,

http://www.ars-grin.gov/). With regard to grapevine, much work has been carried out to investigate the genetic determinants of

skin colour in V. vinifera germplasm collections (This et al.,

Table 1. (Continued )

Cv Clone State Berry description Tissue

code

SV SV SNP VVN VVN VvMybA1 VVN SNP VVN SNP VVI SNP VMC

MUT L he hd hd hd hd Gret/– hd hd hd hd hd hd hd

MUT Wholly mutated buds S he hd hd hd hd Gret/– hd hd hd hd hd hd hd MUT Wholly mutated buds F he hd hd hd hd Gret/– hd hd hd hd hd hd hd

PB 72FR WT L he he he hd hd Gret/– he he he he he he he

WT White-skinned berries S he he he hd hd Gret/– he he he he he he he WT White-skinned berries F he he he hd hd Gret/– he he he he he he he

PB CL55 WT L he he he hd hd Gret/– he he he he he he he

PB CL54 WT L he he he hd hd Gret/– he he he he he he he

PB 33W WT L he he he hd hd Gret/– he he he he he he he

PB 209D WT L he he he hd hd Gret/– he he he he he he he

PB 212D WT L he he he hd hd Gret/– he he he he he he he

PB VCR5 WT L he he he hd hd Gret/– he he he he he he he

PB VCR7 WT L he he he hd hd Gret/– he he he he he he he

WT White-skinned berries S he he he hd hd Gret/– he he he he he he he WT White-skinned berries F he he he hd hd Gret/– he he he he he he he L, leaf; S, berry skin; F, berry flesh; R, root; he, heterozygous genetic state; hd, heterozygous deletion.

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2007; Walker et al., 2007; Fournier-Level et al., 2009, 2010), as

well as in Vitis species and hybrids (Cadle-Davidson et al., 2008;

Azuma et al., 2008, 2011; Shimazaki et al., 2011). However, the berry colour locus has been less explored in somatic variants of a

grape variety (cépage, family of cultivars; Boursiquot and This,

1999), there being two reports, namely Giannetto et al. (2008)

and Walker et al. (2006). In this latter study, somatic mutations of Cabernet Sauvignon, Malian, and Shalistin have been charac-terized at the molecular level, resulting in the development of the first evolutionary model (here reported as the ‘CabSau-model’) that explains the development of somatic colour mutants within a

variety (Walker et al., 2006). Malian bearing bronze-skinned

ber-ries is a periclinal chimera of Cabernet Sauvignon, and Shalistin bearing white-skinned berries is a bud sport of Malian. The bronze berry phenotype of Malian is related to a large (>260 kb) deletion in the colour locus, which includes the functional allele of VvMybA1 and VvMybA2 in the L2 cells, while the white berry phenotype of Shalistin is the result of a cellular rearrangement (or displacement) in Malian whereby the L2 cell layer (unpig-mented phenotype) replaces the L1 cells (pig(unpig-mented phenotype) (Walker et al., 2006).

The structural dynamics at the berry colour locus

In Pinot gris the findings revealed a very large heterozygous dele-tion of 4202–4350 kb that represents a relevant structural change affecting about a quarter of chromosome 2 in the L2 cells. In addition to the VvMybA1 functional allele, this deletion encom-passes one allele of 194 genes according to the current 12X grape

reference genome annotation (http://www.genoscope.cns.fr). As

the grape sex locus (Dalbò et al., 2000; Marguerit et al., 2009)

was mapped ~10 Mb upstream to the 5’ border of the deletion, it is relevant to emphasize that the occurrence of the mutation in the L2 cells, which gives rise to the gametes, did not prevent the current use of Pinot gris as a parental genotype for sexual propa-gation and thus in breeding programmes.

The present comprehensive study, in agreement with the

first suggestion by Hocquigny et al. (2004), builds on the

model suggested by Walker et al. (2006) that envisaged a large

deletion-similar to the one in Malian-in the L2 of Pinot gris, based on a non-layer-specific cleaved amplified polymorphic sequences (CAPS) analysis. In addition to the layer localization of this deletion, the phenotype difference between Pinot gris and Pinot noir is probably the result of a displacement of L2 cells (unpigmented phenotype) into L1 cells (pigmented phe-notype), as suggested by the different height of the peaks of the electropherograms in the L1+L2-derived tissues of Pinot gris (Fig. 2) and as proposed for Malian and Pinot gris by Walker

et al. (2006).

Furthermore, the present results show that Pinot blanc har-bours a short heterozygous deletion, in the 100–179 kb range, encompassing both the VvMybA1 and VvMybA2 genes. As has occurred in Pinot gris, the deletion affects the VvMybA1 func-tional allele. However, unlike Pinot gris, this deletion is present in both L1 and L2 cells in Pinot blanc. A partially deleted func-tional VvMybA1 allele was suggested to exist in Pinot blanc by

Yakushiji et al. (2006) based on a non-layer-specific Southern blot approach. Since the SAM is organized in separate layers, it

is likely that the deletion affected an L2 cell, which subsequently multiplied, colonizing the L1 cells.

In conclusion, both Pinot gris and Pinot blanc bear a mutation causing loss of function.

The distinctiveness of Pinot gris bud sports and Pinot blanc

In this study, by scoring the plants of Pinot gris clones in the field, partially or wholly white-skinned berries (derived chime-ras or sports) were observed with a frequency range of 1–19% for six of these clones (2010; data not shown). As expected, the chimeric instability was higher in the non-registered than in the registered clones. Considering that this phenomenon is affected

by changes in the environment (Whitham et al., 1981), the

occurrence of these Pinot gris-derived chimeras and sports is in agreement with what was previously described in the literature (Hocquigny et al., 2004; Furiya et al., 2008; Pelsy, 2010). In fact, their generation can occur as a consequence of a rearrange-ment (or displacerearrange-ment) of cell layers in an original chimera,

being itself unstable (Dermen, 1960; Bergann, 1967). In the case

of loss of a functional allele, as in Pinot gris, this mechanism is expected to generate dominant phenotypes in chimeric sec-tors that can be readily exposed to natural or anthropic selection (Fernandez et al., 2010). Such a mosaic of genetic variation, that more probably exists in plants that have multiple apical mer-istems and/or exhibit a propensity for clonal propagation (e.g. many grasses, shrubs, and fruit trees-such as grape) than in plants that exhibit a high degree of apical dominance and rarely clone (e.g. many conifers), implies that there is a potential ecological

and adaptive significance (Whitham et al., 1981) and represents

an untapped resource for investigations of cell interactions

dur-ing plant development (e.g. Boss and Thomas, 2002).

In order to ascertain the complete cell displacement in the Pinot gris chimeras and bud sports and to exclude the hypothesis that Pinot blanc is a bud sport of Pinot gris, the layer-specific genetic analysis was extended to the Pinot gris-derived mutants and to additional Pinot blanc clones. The results revealed the presence of the 4202–4350 kb deletion in all white skin sectors and wholly mutated skins of Pinot gris, confirming that these cells hold an L2 genetic origin, carrying the information for the

unpigmented phenotype (Hocquigny et al., 2004). Colourless

sectors of Pinot gris are analogous to the hairless sectors of Pinot meunier in which cells from the L2 layer displace cells of the L1

layer (Stenkamp et al., 2009). The finding are, indeed, in

con-trast to the study performed by Furiya et al. (2008), relying on

one triallelic SSR profile based on a non-layer-specific survey in Pinot gris white-skinned berries. With regards to the additional Pinot blanc clones, they showed, at the cell layer level, the same genetic profile, which includes the 100–179 kb deletion, as the Pinot blanc clones belonging to the core set. This result demon-strated that the Pinot gris clones and the Pinot blanc clones do not possess the same deletion pattern, suggesting that they have arisen from two independent ancestral mutations.

Besides deletions, structural changes can be caused by point mutations, duplications, inversions, aneuploidy, polyploidy, and

extrachromosomal mechanisms of inheritance (Hartmann and

Kester, 1975). Genomic loss can be retrotrasposition driven, and

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the rate at which transposon-induced mutations arise appears to be affected by the stress or shock experienced by the genome (McClintock, 1984). It seems probable that virus infection, very common in plants, may be among the most important natu-ral stresses responsible for transposon activation. As recently

observed in other Pinot noir clones (Carrier et al., 2012), it can

be supposed that the structural changes that occurred in Pinot gris and in Pinot blanc were stress mediated, resulting in the acti-vation of a mobile genetic element. It is known that

retrotranspo-sons (class I; Wicker et al., 2007) contribute to genome dynamics

and to structural diversity in different plant species (Vitte and

Bennetzen, 2006). In fact, unequal intrastrand (or illegitimate) recombination between the two long terminal repeats (LTRs), that terminate LTR retrotransposons, often generates solo LTRs, with the associated loss of one LTR and the internal sequences of

the element (Roeder and Fink, 1980). Therefore, unequal

recom-bination between homologous LTR retrotransposons at different genomic locations can cause large and net deletion (or

duplica-tion) of nuclear DNA between the elements (Garfinkel, 2005),

similar to those detected here in Pinot gris and Pinot blanc.

The evolutionary model of Pinots

Based on the overall experimental data, in particular on the rel-evant difference in the size of the chromosome deletion, it is suggested that: (i) Pinot blanc is not a bud sport of Pinot gris; and (ii) Pinot blanc and Pinot gris arose as independent somatic mutations of Pinot noir.

Theoretically, the origin of a colourless berry mutant can be ascribed to two distinct models: (i) the sequential model, where the black-skinned berry ancestor gave rise to the grey-skinned which in turn gave rise to the white-skinned berry mutant; and (ii) the parallel model, where the black-skinned berry ances-tor gave rise to the grey-skinned and the white-skinned berry mutants separately.

Here, the parallel model is proposed as the evolutionary model

for the formation of Pinot berry colour somatic variants (Fig. 4).

According to this novel model, the somatic mutants Pinot gris and Pinot blanc arose from the ancestral Pinot noir cultivar inde-pendently. This parallel model is named the ‘Pinot-model’, dis-tinct from the previously reported sequential ‘CabSau-model’ (Walker et al., 2006). Moreover, these results elucidated the rela-tionship between Pinot blanc and Pinot gris. Finally, the name Pinot verdâtre is suggested for the unpigmented bud sport of Pinot gris, holding a peculiar genetic make-up and a green-like phenotype.

These findings represent a breakthrough towards the full understanding of the mechanisms behind the formation of white, grey, red, pink, and tintoreous grape cultivars, the overall pheno-types of which determine a specific enological aptitude.

Supplementary data

Supplementary data are available at JXB online.

Table S1. List of the studied Pinots.

Fig. 4. Model of the formation of Pinot blanc, Pinot gris, and Pinot verdâtre from Pinot noir. A scheme represents the structural dynamics at the berry colour locus in the L1 and in the L2 of Pinot noir, Pinot blanc, Pinot gris, and Pinot verdâtre.

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Table S2. Primer information of the microsatellite markers used.

Table S3. Primer information of the studied regions distrib-uted along chromosome 2.

Table S4. Genetic profile of the Pinot core set at the genome level.

Table S5. Genetic make-up of the Pinot core set at the berry colour locus.

Table S6. Genotypic state of the Pinot core set at all the ana-lysed loci along chromosome 2.

Acknowledgements

The authors are grateful to L. Bianchedi (FEM, IT) for sup-port in sample preparation, to A. Ciccotti (FEM, IT) for the in

vitro maintenance of the IASMA grape clone collection, and

to J. Terleth (Laimburg, IT) and L. Bavaresco (CRA-VIT, IT) for providing additional grape clone materials. The authors also wish to thank the Sequencing and Genotyping Platform team (FEM, IT) for helpful suggestions on SNPlex assay. We grate-fully acknowledge E. De Paoli for helpful discussion. Finally, we wish to thank P.K. Boss for proofreading and critically reading the manuscript, and the anonymous reviewers for their useful suggestions. This work was supported by the S.A.M.P.LE. Vitis post-doc project funded by the Autonomous Province of Trento.

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